Project Summary/Abstract Clustered DNA damage is a unique class of DNA lesion generated by ionizing radiation. This class of DNA lesion consists of multiple DNA damage in close proximity to each other. Inappropriate processing of clustered lesion by cellular processes can lead to increased lethality and genomic instability in cells. Tandem base damage is a simple member of clustered lesion. Tandem lesions are processed via two major DNA repair pathways, the base excision and nucleotide excision repair pathway. However, incomplete repair of tandem base lesion occurs when the lesion is processed by DNA glycosylases, members of the base excision repair pathway. These data suggest that other DNA repair enzymes or factors are crucial for the subsequent complete repair of tandem base lesions. Saccharomyces cerevisiae or Baker [unreadable]s yeast is a powerful model system for understanding the in vivo processing of tandem lesions. We have adopted and modified the single strand oligonucleotide transformation assay in yeast, allowing us to systematically examine the mechanism by which yeast process tandem lesions. Tandem lesions are introduced into yeast chromosomal DNA by transformation using single stranded oligonucleotide containing unique tandem lesion. Three aims are included in this application. The first aim is directed to examine the mechanism and pathway by which tandem lesions are repaired. The second aim is devoted to determine the mutagenic potential of unrepaired tandem lesion. The third aim is directed to investigate the role of DNA polymerases and other DNA repair factors that are essential for the removal of remaining DNA lesion after the incomplete processing of tandem lesion by DNA glycosylases. Completion of these three aims is expected to provide significant understanding on how cells process tandem lesions and the biological consequences of incompletely repaired tandem lesions.